Pre-therapy Plasma Research Articles

Abstract 5172: miRNA lncRNA network associated with response to cemiplimab in cutaneous squamous cell carcinoma

Abstract Introduction: For advanced cSCC the use of immunological therapies relies on the biology of the tumor, characterized by high mutational burden. However, although the anti-PD1 cemiplimab showed to be a promising treatment option, future research focusing on the use of biomarkers to predict treatment response are needed. Since increasing number of studies has revealed the crucial roles of non-coding RNAs, such as miRNAs and lncRNAs in the development of SCC, the present study had the purpose to explore the lncRNA-miRNA-mRNA networks associated with response to anti-PD1. For this purpose the experimental procedure envisaged a preliminary in silico analysis followed by the investigation of identified miRNA and lncDNA in liquid biopsy. Methods: For the in silico study, GSE139505 RNA-sequencing raw data have been retrieved from SRA, including 7 Healthy Samples and 9 cSCC biopsies. Two matrices have been prepared, including coding genes and lncRNAs. Normalized coding gene matrix have been used as input to perform GSEA. Coexpression analysis between lncRNAs and Core enrichment genes resulting from GSEA was performed. Biological network including validated miRNA-gene interactions (TarBase) and lncRNA-miRNA interactions (lncBase) has been built-up through Cytoscape. MCODE analysis to identify subnetwork with the closest relationships was performed. The quantification of identified miRNAs (miR-125a, miR-125b, miR-148a, miR-148b) and lnc (AF131217.1, RP11-362F19.1, PVT1, LINC00665) was performed in pre-therapy plasma samples of 34 cSCC patients, categorized according to clinical response to cemiplimab, in responders and non-responders. miRNA-specific RT primers were used for reverse transcription by ddPCR. Results: The computational analysis revealed a significant enrichment of two gene sets related to immunotherapy, namely the Reactome_PD1_signaling and WP_Cancer_Immunotherapy_by_PD1_blockade. The coespression analyses highlighted more than 600 correlations gene-miRNA-lncRNA for the two above-mentioned genesets. CD4-miR-125a/b and AF131217.1/RP11-362F19.1 are strongly correlated for the first one; whilst, regarding the latter one, CD274/HLA-A with mir-148a/b and PVT1/LINC00665 and JUN with mir-125a/b and AF131217.1/RP11-362F19.1 were found. The validation in cSCC plasma samples evidenced the significant correlation between mir-148a/b and PVT1/LINC00665 and an higher expression of all miRNAs in responders respect to non-responders. The longitudinal study assessing the correlation between miRNA expression and response to cemiplimab is ongoing. Conclusion: Preliminary analysis in pre-therapy plasma samples of cSCC validated the in silico data highlighting the inverse correlation between miR-148a/b and lnc (PVT1 and LINC00665). Further investigation will confirm the role of miR125a/b and miR148a/b as predictors of cemiplimab response. Citation Format: Letizia Porcelli, Simona De Summa, Rossella Fasano, Michele Guida, Tommaso M. Marvulli, Tania Rafaschieri, Giuseppe De Palma, Simona Serratì, Roberta Di Fonte, Ivana De Risi, Sabino Strippoli, Stefania Tommasi, Amalia Azzariti. miRNA lncRNA network associated with response to cemiplimab in cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5172.

Circulating extracellular vesicles are monitoring biomarkers of anti-PD1 response and enhancer of tumor progression and immunosuppression in metastatic melanoma

BackgroundClinical drawback in checkpoint inhibitors immunotherapy (ICI) of metastatic melanoma (MM) is monitoring clinical benefit. Soluble forms of PD1(sPD1) and PD-L1(sPD-L1) and extracellular vesicles (EVs) expressing PD1 and PD-L1 have recently emerged as predictive biomarkers of response. As factors released in the blood, EVs and soluble forms could be relevant in monitoring treatment efficacy and adaptive resistance to ICI.MethodsWe used pre-therapy plasma samples of 110 MM patients and longitudinal samples of 46 patients. Elisa assay and flow cytometry (FCM) were used to measure sPD-L1 and sPD1 concentrations and the percentage of PD1+ EVs and PD-L1+ EVs, released from tumor and immune cells in patients subsets. Transwell assays were conducted to investigate the impact of EVs of each patient subset on MM cells invasion and interaction between tumor cells and macrophages or dendritic cells. Viability assays were performed to assess EVs effect on MM cells and organoids sensitivity to anti-PD1. FCM was used to investigate immunosuppressive markers in EVs and immune cells.ResultsThe concentrations of sPD1 and sPD-L1 in pre-treatment and longitudinal samples did not correlate with anti-PD1 response, instead only tumor-derived PD1+ EVs decreased in long responders while increased during disease progression in responders. Notably, we observed reduction of T cell derived EVs expressing LAG3+ and PD1+ in long responders and their increase in responders experiencing progression. By investigating the impact of EVs on disease progression, we found that those isolated from non-responders and from patients with progression disease accelerated tumor cells invasiveness and migration towards macrophages, while EVs of long responders reduced the metastatic potential of MM cells and neo-angiogenesis. Additionally, the EVs of non-responders and of progression disease patients subset reduced the sensitivity of MM cells and organoids of responder to anti-PD1 and the recruitment of dendritic cells, while the EVs of progression disease subset skewed macrophages to express higher level of PDL-1.ConclusionCollectively, we suggest that the detection of tumor-derived PD1 + EVs may represent a useful tool for monitoring the response to anti-PD1 and a role for EVs shed by tumor and immune cells in promoting tumor progression and immune dysfunction.Graphical

Survival prediction optimization of acute myeloid leukaemia based on T-cell function-related genes and plasma proteins.

Interactions between acute myeloid leukaemia (AML) cells and immune cells are postulated to corelate with outcomes of AML patients. However, data on T-cell function-related signature are not included in current AML survival prognosis models. We examined data of RNA matrices from 1611 persons with AML extracted from public databases arrayed in a training and three validation cohorts. We developed an eight-gene T-cell function-related signature using the random survival forest variable hunting algorithm. Accuracy of gene identification was tested in a real-world cohort by quantifying cognate plasma protein concentrations. The model had robust prognostic accuracy in the training and validation cohorts with five-year areas under receiver-operator characteristic curve (AUROC) of 0.67-0.76. The signature was divided into high- and low-risk scores using an optimum cut-off value. Five-year survival in the high-risk groups was 6%-23% compared with 42%-58% in the low-risk groups in all the cohorts (all p values <0.001). In multivariable analyses, a high-risk score independently predicted briefer survival with hazard ratios of death in the range 1.28-2.59. Gene set enrichment analyses indicated significant enrichment for genes involved in immune suppression pathways in the high-risk groups. Accuracy of the gene signature was validated in a real-world cohort with 88 pretherapy plasma samples. In scRNA-seq analyses most genes in the signature were transcribed in leukaemia cells. Combining the gene expression signature with the 2017 European LeukemiaNet classification significantly increased survival prediction accuracy with a five-year AUROC of 0.82 compared with 0.76 (p < 0.001). Our T-cell function-related risk score complements current AML prognosis models.

Limited Sequence Variation and Similar Phenotypic Characteristics of HIV-1 Subtype C Gag Variants Derived From the Reservoir and Pre-Therapy Plasma

HIV variants present in the reservoir, particularly in tissues, may differ from those present in peripheral blood prior to therapy initiation, and characterisation of these reservoir variants could better inform immune-based interventions for HIV cure. In the present study, Gag sequence differences between variants derived from the lymph node and peripheral blood mononuclear cell (PBMC) reservoirs as well as those derived from pre-therapy plasma, were investigated in 24 HIV-1 subtype C-infected individuals. HIVgagamplification was successful for 20 individuals, where 4 were controls including one untreated individual and 3 early treated individuals with LN collection within 2 weeks of treatment initiation. The remaining 16 individuals with LN and PBMC collection > 3 months after treatment initiation (median = 665 days), were further characterised. Recombinant viruses encoding patient-derived Gag-protease sequences from the pre-therapy plasma, LN reservoir, and PBMC reservoir, were constructed and the replication-competent viruses that grewin vitrowere used to further investigate whether there are specific features of Gag reservoir variants that may have relevance for strategies to cure HIV. Virus characteristics measured included replication capacity, interferon-alpha resistance, cell-to-cell spread ability, and induction of antiviral cytokines. A limited number of novel Gag mutations (median = 4) in the reservoir of 3/7 early treated participants and 9/9 late treated participants were observed, where the majority of these mutations were likely cytotoxic T lymphocyte (CTL)-driven and 48% were represented in the replication-competent viruses. The reservoir variants had very few unique potential CTL escape mutations (median = 3) in Gag compared to the number of these Gag mutations that were already present in the plasma-derived virus (median = 23) at the time of treatment initiation, which was similar whether treatment was initiated late or early. The data suggest that the extent of CTL escape in Gag overall is likely similar between early and late treated individuals as well as between the reservoir and pre-therapy variants. The sequence differences in Gag that were unique to the reservoir viruses did not result in significantly altered virus characteristics overall, and are therefore unlikely to affect effectiveness of immune-based interventions for virus eradication.

Small Extracellular Vesicles in Pre-Therapy Plasma Predict Clinical Outcome in Non-Small-Cell Lung Cancer Patients.

Simple SummarySince re-biopsy of the primary tumor or metastatic lesions is not an option in most NSCLC patients, the use of the “liquid tumor biopsy” represents a promising non-invasive diagnostic/prognostic approach. Small extracellular vesicles (sEV) are currently emerging as a promising source for liquid tumor biopsies. The present study reports that pre-therapy total sEV protein (TEP) levels are associated with patient clinical outcome. We show that the high sEV TEP levels in pre-treatment plasma are a significant negative predictor of PFS and OS in treatment-naïve NSCLC patients. We also observed a positive correlation between the sEV TEP levels and the percentages of circulating CD8+PD-1+ and CD8+PD-L1+T cells at baseline prior to any therapy. This suggested that sEV in plasma hinders anti-tumor immune responses via the PD-L1/PD-1 pathway. The data suggest that pre-therapy plasma sEV levels could be useful as non-invasive predictors of NSCLC progression and outcome after conventional therapy.The potential use of plasma-derived small extracellular vesicles (sEV) as predictors of response to therapy and clinical outcome in chemotherapy-naïve patients with non-small-cell lung cancer (NSCLC) was explored. sEV were isolated by size-exclusion chromatography from the plasma of 79 chemotherapy-naïve NSCLC patients and 12 healthy donors (HD). sEV were characterized with regard to protein content, particle size, counts by qNano, morphology by transmission electron microscopy, and molecular profiles by Western blots. PD-1 and PD-L1 expression on circulating immune cells was analysed by flow cytometry. Pre-treatment levels of total sEV protein (TEP) were correlated with overall (OS) and progression-free survival (PFS). The sEV numbers and protein levels were significantly elevated in the plasma of NSCLC patients compared to HD (p = 0.009 and 0.0001, respectively). Baseline TEP levels were higher in patients who developed progressive disease compared to patients with stable disease (p = 0.007 and 0.001, stage III and IV, respectively). Patient-derived sEV were enriched in immunosuppressive proteins as compared to proteins carried by sEV from HD. TEP levels were positively correlated with CD8+PD-1+ and CD8+PD-L1+ circulating T cell percentages and were independently associated with poorer PFS (p < 0.00001) and OS (p < 0.00001). Pre-therapy sEV could be useful as non-invasive biomarkers of response to therapy and clinical outcome in NSCLC.

Defining the Inflammatory Plasma Proteome in Pediatric Hodgkin Lymphoma.

Simple SummaryHodgkin lymphoma (HL) is a common type of cancer that is characterized by rare, malignant cells among an inflammatory microenvironment. Specific systemic, inflammatory plasma proteins have demonstrated prognostic significance in adult HL; however, systemic inflammation has not been well-characterized in childhood HL. The aim of our study was to better define the inflammatory pre-therapy plasma proteome and identify plasma proteins associated with clinical features of childhood HL. We measured plasma concentrations of 135 proteins in 56 pediatric subjects with newly diagnosed HL and 47 healthy pediatric controls. We found that the plasma protein profile was distinct from controls, and unique proteins were associated with high-risk disease (IL-10, TNF-α, IFN-γ, IL-8), slow early therapy response (CCL13, IFN-λ1, IL-8), and relapse (TNFSF10). These proteins could be used to improve risk stratification, and thus optimize outcomes and minimize unnecessary toxic exposures for those with childhood HL.Hodgkin lymphoma (HL) histopathology is characterized by rare malignant Reed–Sternberg cells among an inflammatory infiltrate. We hypothesized that characteristics of inflammation in pediatric HL lesions would be reflected by the levels of inflammatory cytokines or chemokines in pre-therapy plasma of children with HL. The study objectives were to better define the inflammatory pre-therapy plasma proteome and identify plasma biomarkers associated with extent of disease and clinical outcomes in pediatric HL. Pre-therapy plasma samples were obtained from pediatric subjects with newly diagnosed HL and healthy pediatric controls. Plasma concentrations of 135 cytokines/chemokines were measured with the Luminex platform. Associations between protein concentration and disease characteristics were determined using multivariate permutation tests with false discovery control. Fifty-six subjects with HL (mean age: 13 years, range 3–18) and 47 controls were analyzed. The cytokine/chemokine profiles of subjects with HL were distinct from controls, and unique cytokines/chemokines were associated with high-risk disease (IL-10, TNF-α, IFN-γ, IL-8) and slow early response (CCL13, IFN-λ1, IL-8). TNFSF10 was significantly elevated among those who ultimately relapsed and was significantly associated with worse event-free survival. These biomarkers could be incorporated into biologically based risk stratification to optimize outcomes and minimize toxicities in pediatric HL.

Abstract 3147A: The use of the sTRA glycan for predicting responses to neoadjuvant therapy in patients with pancreatic ductal adenocarcinoma

Abstract The sTRA glycan has been validated as a serological and cell-surface biomarker of pancreatic ductal adenocarcinoma (PDAC). In previous work, a biomarker panel using sTRA and CA19-9 performed significantly better than CA19-9 for distinguishing PDAC from benign diseases of the pancreas; here we explored whether the subgroup identified by sTRA has a different prognosis than other subgroups. We examined the plasma levels of sTRA and CA19-9 in two cohorts of patients with resectable or borderline-resectable PDAC. In each cohort, two samples per patient were collected: one prior to any therapy, and another after neoadjuvant therapy but before surgery. In the first cohort (n = 54), the ratio of post-therapy to pre-therapy plasma sTRA was statistically higher (p &amp;lt; 0.05, student’s t-test using logged ratios) in patients with poor response, as assessed by surgical pathology. CA19-9 had higher ratios in a subset of the poor-response subjects but without statistical significance. In the second cohort (n = 75), the pre-treatment and post-treatment levels of sTRA were significantly higher (p &amp;lt; 0.01, Wilcoxon Rank-Sum test) in patients with shorter survival (&amp;lt;2 years). In contrast, CA19-9 did not show statistical significance in the pre-treatment or post-treatment levels, but the post:pre ratios were substantially higher in a subset of the short-survival group. In both cohorts, sTRA identified a subset of poor responders that was non-identical to the one identified by CA19-9. As a result, a biomarker panel combining the markers provided better accuracy for identifying poor responders than either marker alone. In an analysis of 24 cell line models of PDAC, the cell lines that secreted sTRA had a statistically higher resistance to chemotherapy than those not secreting sTRA, whereas the cell lines secreting CA19-9 showed no such association. Therefore, pancreatic cancers that produce sTRA may be a distinct subtype with higher resistance to chemotherapy, and sTRA in combination with CA19-9 could be a valuable serological predictor of responsiveness to chemotherapy. Citation Format: Ben Staal, Ying Liu, Peter Hsueh, Mohammed Aldakkak, Herbert Zeh, Douglas Evans, Randall E. Brand, Susan Tsai, Brian B. Haab. The use of the sTRA glycan for predicting responses to neoadjuvant therapy in patients with pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3147A.

Plasma AR status and cabazitaxel in heavily treated metastatic castration-resistant prostate cancer

BackgroundPlasma androgen receptor (AR) copy number status has been identified as a potential biomarker of response in patients with metastatic castration-resistant prostate cancer (mCRPC) receiving docetaxel or the AR-targeted therapies abiraterone or enzalutamide. However, the relevance of plasma AR status in the context of cabazitaxel therapy is unknown. Patients and methodsBetween September 2011 and January 2018, pretherapy plasma samples were collected from 155 patients treated with second- or third-line cabazitaxel at standard or reduced dose in different biomarker protocols. Droplet digital polymerase chain reaction was used to identify plasma AR gain and normal samples. The primary objective was to evaluate associations of plasma AR status with treatment outcome. In an exploratory analysis, a comparison between plasma AR and treatment type was investigated by incorporating updated data from our prior study of 85 post-docetaxel patients receiving abiraterone or enzalutamide. ResultsWe observed a shorter median overall survival (OS) and progression-free survival (PFS) in AR-gained compared to AR-normal patients (OS 10.5 versus 14.1 months, hazard ratio (HR) = 1.44, 95% confidence interval [CI] 0.98–2.13, P = 0.064 and PFS 4.0 versus 5.0 months, HR = 1.47, 95% CI 1.05–2.07, P = 0.024). In patients with mCRPC receiving second-line therapies, a significant treatment interaction was observed between plasma AR and cabazitaxel versus AR-directed therapies for OS (P = 0.041) but not PFS (P = 0.244). In an exploratory analysis, AR-gained patients treated with initial reduced dose of cabazitaxel had a significantly shorter median OS (7.3 versus 11.5 months, HR = 1.95, 95% CI 1.13–3.38, P = 0.016) and PFS (2.7 versus 5.0 months, HR = 2.27, 95% CI 1.39–3.71, P = 0.001). ConclusionPlasma AR status has a potential clinical utility in patients being considered for cabazitaxel. Validation of these findings in prospective trials is warranted.

Plasma AR status and cabazitaxel in heavily-treated metastatic castration-resistant prostate cancer (mCRPC).

203 Background: Plasma androgen receptor ( AR) copy number status has been identified as a potential biomarker of response in mCRPC patients receiving docetaxel or the AR-targeted therapies abiraterone or enzalutamide. However, the relevance of plasma AR status in the context of cabazitaxel therapy is unknown. Methods: Between September 2011 and January 2018, pre-therapy plasma samples were collected from 155 patients treated with second or third-line cabazitaxel at standard or reduced dose in different biomarker protocols. Droplet digital PCR was used to identify plasma AR gain and normal samples, with the primary objective to evaluate associations of plasma AR status with treatment outcome. In an exploratory analysis, a comparison between plasma AR status and treatment type was investigated by incorporating updated data from our prior study of 85 post-docetaxel patients receiving abiraterone or enzalutamide. Results: We observed a shorter median overall/progression-free survival (OS/PFS) in AR-gained compared to AR-normal patients (OS 10.5 versus 14.1 months, hazard ratio (HR) 1.44, 95% confidence interval (CI) 0.98-2.13, P = 0.064), and (PFS 4.0 versus 5.0 months, HR 1.47, 95%CI 1.05-2.07, P = 0.024). In mCRPC patients receiving second-line therapies, a significant treatment interaction was observed between plasma AR and cabazitaxel versus AR-directed therapies for OS (P = 0.041) but not PFS (P = 0.244). In an exploratory analysis, AR-gained patients treated with initial reduced-dose of cabazitaxel had a significantly shorter median OS (7.3 versus 11.5 months, HR 1.95, 95%CI 1.13-3.38, P = 0.016), and PFS (2.7 versus 5.0 months, HR 2.27, 95%CI 1.39-3.71, P = 0.001). Conclusions: Plasma AR status has a potential clinical utility in patients being considered for cabazitaxel. Validation of these findings in prospective trials are warranted.

Circulating exosomes carrying an immunosuppressive cargo interfere with cellular immunotherapy in acute myeloid leukemia

Exosomes, small (30–150 nm) extracellular vesicles (EVs) isolated from plasma of patients with acute myeloid leukemia (AML) carry leukemia-associated antigens and multiple inhibitory molecules. Circulating exosomes can deliver suppressive cargos to immune recipient cells, inhibiting anti-tumor activities. Pre-therapy plasma of refractory/relapsed AML patients contains elevated levels of immunosuppressive exosomes which interfere with anti-leukemia functions of activated immune cells. We show that exosomes isolated from pre-therapy plasma of the AML patients receiving adoptive NK-92 cell therapy block anti-leukemia cytotoxicity of NK-92 cells and other NK-92 cell functions. NK-92 cells do not internalize AML exosomes. Instead, signaling via surface receptors expressed on NK-92 cells, AML exosomes simultaneously deliver multiple inhibitory ligands to the cognate receptors. The signals are processed downstream and activate multiple suppressive pathways in NK-92 cells. AML exosomes reprogram NK-92 cells, interfering with their anti-leukemia functions and reducing the therapeutic potential of adoptive cell transfers. Plasma-derived exosomes interfere with immune cells used for adoptive cell therapy and may limit expected therapeutic benefits of adoptive cell therapy.

Abstract P3-03-03: An acquired HER2 T798I gatekeeper mutation induces resistance to neratinib in a patient with HER2 mutant-driven breast cancer

Abstract ERBB2, the gene encoding HER2, is mutated in 2-4% of breast cancers. The HER2 irreversible tyrosine kinase inhibitor (TKI) neratinib has shown clinical activity against breast cancer cells harboring HER2 activating mutations. Here, we report for the first time an acquired gatekeeper HER2T798I mutation in a patient with HER2-mutant breast cancer after an initial exceptional response to neratinib. A patient with ER+/PR+/HER2-negative invasive lobular breast cancer progressing on standard therapy was found to harbor a L869R kinase domain mutation in HER2. HER2L869R is homologous to the known activating mutation EGFRL861R/Q. MCF10A breast epithelial cells expressing HER2L869R displayed enhanced HER2-mediated signaling and were resistant to lapatinib and trastuzumab but sensitive to neratinib. The patient was enrolled in the phase II SUMMIT trial (NCT01953926) and treated with neratinib, achieving a partial response lasting 16 months before developing progression. Next gen sequencing of DNA from both a new skin metastasis and plasma cell-free DNA (cfDNA) identified HER2L869R (8.7% cfDNA), whereas a novel HER2T798I mutation was detected only in plasma at 1.3%. Deep sequencing of pre-therapy tumor tissue and plasma did not detect HER2T798I, suggesting that this mutation arose upon resistance. HER2T798I has not been reported in TCGA, COSMIC, or among plasma samples from 17,345 cancer patients subjected to digital DNA sequencing using the Guardant360 assay. HER2T798I is homologous to the EGFRT790M, KITT670I and BCR-ABLT315I gatekeeper mutations known to mediate resistance to erlotinib/gefitinib and imatinib. To examine if HER2T798I mediates resistance to neratinib, we employed biochemical and biological assays and molecular modeling of wild-type (WT) HER2 and HER2T798I. Structural modeling showed the increased bulk of the isoleucine at position 798 would result in a steric clash with neratinib, thus reducing drug binding. We stably expressed HER2WT, HER2T798I, HER2L869R and HER2L869R/T798I in MCF10A cells and NR6 mouse fibroblasts. Neratinib (10-100 nM) blocked HER2-mediated signaling in cells expressing HER2WT or HER2L869R but did not in cells expressing HER2T798I. The EGFR irreversible TKI osimertinib (100 nM), which isselective for mutant EGFR (including EGFRT790M) and approved for treatment of NSCLC expressing EGFRT790M, failed to inhibit HER2WT, HER2L869R or HER2T798I. In contrast, either the EGFR/HER2 irreversible TKI afatinib or AZ5104, a metabolite of osimertinib, strongly blocked signaling induced by HER2WT, HER2L869R or HER2T798I. Cells expressing HER2T798M displayed a significantly higher IC50 to neratinib than cells expressing HER2WT, whereas afatinib or AZ5014 were very active against all cells (IC50&amp;lt;10 nM). Conclusions: The acquisition of a T798I gatekeeper mutation in HER2 upon development of clinical resistance to neratinib in a breast cancer with an initial activating mutation in HER2 strongly suggests that HER2L869R is a driver mutation. We speculate that HER2T798I may arise as a secondary mutation following response to effective HER2 TKIs in other cancers with HER2 activating mutations. Certain irreversible EGFR inhibitors may be effective in patients with HER2-driven breast cancer resistant to neratinib. Citation Format: Hanker AB, Red Brewer M, Sheehan JH, Koch JP, Lanman R, Hyman DM, Cutler, Jr. RE, Lalani AS, Cross D, Lovly CM, Meiler J, Arteaga CL. An acquired HER2 T798I gatekeeper mutation induces resistance to neratinib in a patient with HER2 mutant-driven breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-03.

Non-R5-tropic HIV-1 in subtype A1 and D infections were associated with lower pretherapy CD4+ cell count but not with PI/(N)NRTI therapy outcomes in Mbarara, Uganda.

Previous studies suggest that infection with non-R5-tropic subtype B HIV-1, compared with R5, is associated with a more rapid decline in CD4 cell count, but does not affect PI/(N)NRTI therapy outcome. Here, we explored clinical correlates associated with viral tropism in subtype A1 and D infections. HIV-1 subtype A1 (n = 196) and D (n = 143) pretherapy plasma samples and up to 7.5 years of posttherapy virologic and CD4 data were collected from a cross-sectional cohort in Mbarara, Uganda. Tropism and subtype were inferred using env V3 (geno2pheno) and gp41 (RIP) Sanger sequences. For each subtype, R5 infection was compared with non-R5 in terms of: pretherapy viral load and CD4 cell count (Mann-Whitney tests), and therapy outcomes, including time to virologic suppression, postsuppression virologic rebound, CD4 decline and CD4 recovery (log-rank tests). A 94% of all patients in this study achieved virologic suppression within median 3 months posttherapy. In both subtypes, non-R5 infection was associated with lower pretherapy CD4 cell count (non-R5 vs. R5; A: median 57 vs. 147 cells/μl P = 0.005; D: 80 vs. 128 cells/μl P = 0.006). Multivariable linear regression confirmed that tropism, not subtype nor the interaction between subtype and tropism, was a significant predictor of pretherapy CD4 cell count (P < 0.0001). None of pretherapy viral load, time to virologic suppression, virologic rebound, CD4 decline nor CD4 recovery was significantly different (all P > 0.09). Regardless of HIV-1 subtype or tropism, the majority of patients in this Ugandan cohort responded to therapy, even though non-R5 infection was associated with lower pretherapy CD4 cell count.

Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants.

The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible.

Plasma Epstein Barr viral load in adult-onset Hodgkin lymphoma in South India

Objective/backgroundEpstein Barr Virus (EBV) DNA load is increasingly being used as a noninvasive biomarker for detecting EBV association in lymphomas. Since there is a need of data from India, we undertook to prospectively evaluate plasma EBV DNA load as a marker of EBV association in newly diagnosed adult-onset Hodgkin lymphoma (HL). MethodsEBV DNA was quantified using real-time polymerase chain reaction. In a subset of patients, an assay was validated qualitatively with EBV latent membrane protein-1 (LMP1) immunohistochemistry (IHC). Wherever possible, follow-up plasma samples post three cycles of chemotherapy were obtained. ResultsOver a period of 10months, 33 newly diagnosed adult-onset HL were enrolled in the study. Pretherapy plasma EBV DNA was detectable in ∼49% (16/33) patients (viral loads range, 1.0–51.2×103copies/mL) and undetectable in 30 voluntary blood donors. LMP1 IHC was positive in 56% of cases tested (14/25). Sensitivity and specificity of plasma EBV DNA with respect to LMP1 IHC were 86% and 100%, respectively. Of the eight patients in whom follow-up plasma was available, in five EBV baseline-positive patients EBV load reverted to negative postchemotherapy and corroborated with clinical remission. ConclusionPlasma EBV DNA load estimation may be useful in detecting EBV-association and possibly monitoring the response to therapy in EBV-related HL especially in our country where EBV association of HL is higher than in developed nations.

099 Erythrocyte entrapped thymidine phosphorylase (EE-TP) therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

MNGIE is a fatal, autosomal recessive disorder caused by mutations in TYMP, a nuclear gene encoding thymidine phosphorylase (TP), leading to accumulation of thymidine (dThd) and deoxyuridine (dUrd) in all...

Dosimetric Considerations in Radioimmunotherapy and Systemic Radionuclide Therapies: A Review

Radiopharmaceutical therapy, once touted as the “magic bullet” in radiation oncology, is increasingly being used in the treatment of a variety of malignancies; albeit in later disease stages. With ever-increasing public and medical awareness of radiation effects, radiation dosimetry is becoming more important. Dosimetry allows administration of the maximum tolerated radiation dose to the tumor/organ to be treated but limiting radiation to critical organs. Traditional tumor dosimetry involved acquiring pretherapy planar scans and plasma estimates with a diagnostic dose of intended radiopharmaceuticals. New advancements in single photon emission computed tomography and positron emission tomography systems allow semi-quantitative measurements of radiation dosimetry thus allowing treatments tailored to each individual patient.

CD4-Dependent Characteristics of Coreceptor Use and HIV Type 1 V3 Sequence in a Large Population of Therapy-Naive Individuals

We investigated the associations between coreceptor use, V3 loop sequence, and CD4 count in a cross-sectional analysis of a large cohort of chronically HIV-infected, treatment-naive patients. HIV coreceptor usage was determined in the last pretherapy plasma sample for 977 individuals initiating HAART in British Columbia, Canada using the Monogram Trofile Tropism assay. Relative light unit (RLU) readouts from the Trofile assay, as well as HIV V3 loop sequence data, were examined as a function of baseline CD4 cell count for 953 (97%) samples with both phenotype and genotype data available. Median CCR5 RLUs were high for both R5 and X4-capable samples, while CXCR4 RLUs were orders of magnitude lower for X4 samples (p < 0.001). CCR5 RLUs in R5 samples (N = 799) increased with decreasing CD4 count (p < 0.001), but did not vary with plasma viral load (pVL) (p = 0.74). In X4 samples (N = 178), CCR5 RLUs decreased with decreasing CD4 count (p = 0.046) and decreasing pVL (p = 0.097), while CXCR4 RLUs increased with decreasing pVL (p = 0.0008) but did not vary with CD4 (p = 0.96). RLUs varied with the presence of substitutions at V3 loop positions 11, 25, and 6-8. The prevalence and impact of substitutions at codons 25 and 6-8 were CD4 dependent as was the presence of amino acid mixtures in the V3; substitutions at position 11 were CD4 independent. Assay RLU measures predictably vary with both immunological and virological parameters. The ability to predict X4 virus using genotypic determinants at positions 25 and 6-8 of the V3 loop is CD4 dependent, while position 11 appears to be CD4 independent.

ART Suppresses Plasma HIV-1 RNA to a Stable Set Point Predicted by Pretherapy Viremia

Current antiretroviral therapy is effective in suppressing but not eliminating HIV-1 infection. Understanding the source of viral persistence is essential for developing strategies to eradicate HIV-1 infection. We therefore investigated the level of plasma HIV-1 RNA in patients with viremia suppressed to less than 50–75 copies/ml on standard protease inhibitor- or non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy using a new, real-time PCR-based assay for HIV-1 RNA with a limit of detection of one copy of HIV-1 RNA. Single copy assay results revealed that >80% of patients on initial antiretroviral therapy for 60 wk had persistent viremia of one copy/ml or more with an overall median of 3.1 copies/ml. The level of viremia correlated with pretherapy plasma HIV-1 RNA but not with the specific treatment regimen. Longitudinal studies revealed no significant decline in the level of viremia between 60 and 110 wk of suppressive antiretroviral therapy. These data suggest that the persistent viremia on current antiretroviral therapy is derived, at least in part, from long-lived cells that are infected prior to initiation of therapy.

Plasma RNA as an alternative to cells for monitoring molecular response in patients with chronic myeloid leukemia

Quantitation of BCR-ABL mRNA is emerging as the standard of care to monitor the status of chronic myeloid leukemia (CML). Peripheral blood plasma was analyzed in this study because of previous detection of nucleic acids and proteins from tumor cells in plasma samples. Reverse transcriptase polyemrase chain reaction was used to establish ratios of BCR-ABL:ABL mRNA in peripheral blood cells and plasma, and absolute levels of BCR-ABL mRNA per unit volume of plasma. Samples from 160 CML patients and 180 control individuals without CML were tested. Cells and plasma samples from 93 of the CML patients were re-analyzed 3-12 months after imatinib treatment. Ratios of BCR-ABL:ABL mRNA in paired cell and plasma samples of the 160 CML patients correlated significantly (r=0.83; p<0.001). When results were compared directly using the sign test, the pre-therapy plasma results were significantly different from those from peripheral blood cells (p=0.028), but not bone marrow cells (p=0.119). Absolute levels of BCR-ABL mRNA in plasma strongly correlated with many laboratory characteristics in pre-therapy CML patients. Higher BCR-ABL: ABL ratios were detected in plasma samples at all time points after treatment, although this was significant only at 3 months (p=0.0003). In cases in which results from the assays disagreed, minimal residual disease was detected in plasma samples significantly more frequently than in cell samples (p<0.001). Plasma was a reliable source for monitoring BCR-ABL mRNA levels. Minimal residual disease detection from plasma was more sensitive than from cell samples. Our results suggest that absolute levels of BCR-ABL mRNA per unit volume of plasma may reflect tumor load.

Molecular and Clinical Epidemiology of CXCR4‐Using HIV‐1 in a Large Population of Antiretroviral‐Naive Individuals

We wished to characterize the epidemiological and clinical correlates of CXCR4-using human immunodeficiency virus type 1 (HIV-1) ("X4 variants") in a cross-sectional analysis of a large population of antiretroviral-naive individuals. HIV-1 coreceptor use was determined in the last pretherapy plasma sample for 1191 individuals initiating triple-combination therapy in British Columbia, Canada. Baseline variables investigated included sociodemographic characteristics, plasma viral load (pVL), CD4 cell count, AIDS diagnosis, HIV-1 V3 loop sequence, and human CCR5 Delta 32 genotype. Individuals harboring X4 variants (n = 178 of 979 phenotyped samples; 18.2%) displayed a poorer baseline clinical profile than individuals harboring exclusively CCR5-using HIV-1 ("R5 variants") (median pVL, 175,000 vs. 120,000 copies of HIV-1 RNA/mL [P = .0006]; median CD4 cell count, 110 vs. 290 cells/mm(3) [P < .0001]). Individuals heterozygous for the CCR5 Delta 32 deletion (n = 128 of 967; 13.2%) were at 2.5 times higher risk of harboring X4 variants, compared with those without the deletion (multivariate P = .0005). The presence of basic amino acids at codon 11 and/or codon 25 of HIV-1 V3 (n = 109 of 955; 11.4%) was associated with a 9.1 times higher risk of harboring X4 variants (multivariate P < .0001), regardless of CCR5 Delta 32 genotype. In multivariate analyses adjusting for baseline parameters, HIV-1 coreceptor use was not found to be a significant predictor of survival or treatment response. Baseline CD4 cell count, pVL, HIV-1 V3 sequence, and CCR5 Delta 32 genotype were the strongest determinants of CXCR4-using HIV-1 in this population. After adjustment for baseline parameters, the presence of X4 variants before initiation of highly active antiretroviral therapy was not independently associated with a poorer outcome of therapy.